Abstract
African swine fever virus polymerase X (pol X) is the smallest DNA polymerase known (174 amino acids), and its tertiary structure resembles the C-terminal half of prototypical X-family pol beta, which includes a catalytic dNTP-binding site (palm domain) and a finger domain. This structural similarity and the presence of viral genes coding for other base excision repair proteins suggest that pol X functions in a manner similar to pol beta, but inconsistencies concerning pol X catalysis have been reported. We examined the structural and functional properties of two forms of pol X using spectroscopic and kinetic analysis. Using (1)H-(15)N correlated NMR, we unambiguously demonstrated the slow interconversion of pol X between a reduced (pol X(red)) and an oxidized form (pol X(ox)), confirmed by mass spectrometry. Steady-state kinetic analysis revealed that pol X(ox), with a disulfide bond between Cys-81 and Cys-86, has approximately 10-fold lower fidelity than pol X(red) during dNTP insertion opposite a template G. The disulfide linkage is located between two beta-strands in the palm domain, near the putative dNTP-binding site. Structural alignment of pol X with a pol beta ternary structure suggests that the disulfide switch may modulate fidelity by altering the ability of the palm domain to align and stabilize the primer terminus and catalytic metal ion for deprotonation of the 3'-OH group and subsequent phosphoryl transfer. Thus, DNA polymerase fidelity is altered by the redox state of the enzyme and its related conformational changes.
Highlights
The African swine fever virus (ASFV) genome codes for a number of proteins vitally involved in DNA replication, including a minimal repair system [6], consistent with the later stage replication inside the perinuclear space. polymerase X (pol X), one of the two DNA polymerases encoded in the ASFV genome, is a close structural analogue of the well known mammalian DNA polymerase pol  [27], involved in the BER pathway [28]
Oxidized and Reduced Forms of ASFV pol X—A comparison of various 1H-15N HSQC data collected on pol X indicated that a number of amide resonances were shifting with time (Fig. 2)
The structure of pol X, as reported by Maciejewski et al [36, 37], revealed that the only two cysteines present in pol X are located in the -sheet of the palm domain
Summary
ATP-dependent DNA ligase [6]. No DNA glycosylase has been identified far, and BER would require spontaneous depurination/depyrimidation or strand breaks induced by attacks of reactive oxygen species on the genomic DNA [29]. The two forms are distinguished by the formation of either a disulfide bridge between Cys-81 and Cys-86 or their free thiols Both cysteines reside in the palm domain of pol X and are the only cysteines present in the protein. One structure was described to contain reduced cysteines [36], and the other structure was reported to contain a disulfide linkage [30] The latter structure was based on the sole observation of two downfield-shifted 13C- cysteine resonances. Both of the reported buffer systems contained the reducing agent DTT at 10 mM [36] and 1 mM [30], respectively, which was not sufficient to keep the protein in the reduced form for the second structure. The results reported here expand our understanding of how DNA polymerases facilitate genomic replication and may represent a previously unappreciated aspect of viral mutagenesis
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
